1. Field of the Invention
This invention relates to a method of making an electrophoresis sheet used for separation, analysis or the like of a substance having an electrically dissociated group in a solution, like protein or nucleic acid, on the basis of a difference in the electric charge and molecular weight of the particles.
2. Description of the Prior Art
There has heretofore been known an operation of electrophoresis by which separation of a substance by utilizing migration of charged molecules or particles of protein, nucleic acid, their decomposition products, or the like, is effected in a sheet-like medium such as a gel membrane or a filter paper impregnated with a buffer solution under the effect of an electric field. The electrophoresis is utilized particularly for separation and fixation of the high molecular weight substances of the living body as mentioned above.
Particularly, in the generic engineering which has attracted attention in recent years, the electrophoresis operation is indispensable for determining a base sequence in the molecule of nucleic acid such as DNA by utilizing autoradiography. In general, the electrophoresis operation for this purpose includes the step of subjecting a series of mixtures of base-specific reaction products of DNA or DNA fragments provided with a radioactive label to migration in the electric field inside of an electrophoresis medium, whereby the mixtures migrate in parallel with each other in the direction of the electric field. The migration pattern of multiple rows obtained after the migration (a group of zones formed by electrophoresis on the medium) is recorded as an autoradiograph, and then the base arrangement is determined by comparing positions of the zones in the respective rows with each other. The comparison is carried out based on the electrophoresis principle that base-specific reaction products having equal molecular weights migrate by equal distances if the electrophoresis is started from the same line.
In general, filter paper, a membrane filter, a starch gel membrane, a polyacrylamide gel membrane or the like is used as the electrophoresis medium in the form of a sheet having a uniform thickness. In the case where a gel membrane such as a starch gel membrane or a polyacrylamide gel membrane is used, the liquid for gel preparation has heretofore been introduced into a mold constituted by disposing a supporting frame (spacer) around a flat supporting member formed of a non-conductive material such as a glass plate, and gelled for use as the gel membrane, after the upper surface is enclosed by a different supporting member if necessary. However, this method of forming a gel membrane is very troublesome as it requires complicated operations prior to the electrophoresis operation.
Accordingly, as disclosed in, for example, Japanese Unexamined Patent Publication No. 59(1984)-126237, the applicant proposed an electrophoresis sheet which requires no troublesome operations of gel preparation and is easy to use. The proposed electrophoresis sheet comprises two sheet members formed of a non-conductive organic polymer film and disposed to stand facing each other, spacers having predetermined thicknesses disposed at both edge portions between the two sheet members, and an electrophoresis gel membrane enclosed between the two sheet members in a uniform thickness. With the proposed electrophoresis sheet, since the gel membrane can be supplied in the form disposed between the two polymer films, the operators can purchase the electrophoresis sheets and easily carry out the electrophoresis operation.
The aforesaid electrophoresis sheet is provided with slots for introducing and holding the substance subjected to the electrophoresis at an upper edge portion of the gel membrane as the electrophoresis medium, i.e. at the edge portion which does not stand facing the aforesaid spacers and is positioned on the upper side in the course of the electrophoresis operation. The slots extend from the upper edge portion of the gel membrane toward the center thereof and are ordinarily formed side by side in the transverse direction of the gel membrane. In the case where the gel membrane is formed by pouring the liquid for gel preparation into the space between the flat plate-like supporting members formed of glass plates or the like as mentioned above, a comb-like member generally called a sample comb is disposed between the supporting members before the liquid for gel preparation is poured, and removed after gelling of the liquid, thereby to form the slots. However, in the course of making the aforesaid electrophoresis sheet, since pouring of the liquid for gel preparation is not carried out, but instead a gel membrane web already gelled is supported between the sheet members, the slots are formed by punching or cutting of the gel membrane web. Cutting of the slots has heretofore been carried out by, for example, pushing a cutting blade 70 having a shape as shown in FIG. 9 gainst a gel membrane web 71W. The conventional cutting blade 70 is formed into the slot shape by, for example, electrical discharge machining. However, the cutting edge portion of the cutting blade 70 made in this manner is not sharp, but instead substantially has a thickness. Therefore, as shown in FIG. 10, a gel membrane 71 having slots 72, 72, . . . cut by use of the cutting blade 70 becomes such that a corner 72a of each of the slots 72, 72, . . . is round and a closed edge portion 72b has a collapsed rough surface instead of a sharp cut surface. As a result, a substance 73 introduced and held in the slots 72, 72, . . . for electrophoresis becomes a dumpling-like form, and the obtained migration pattern is distorted and cannot be discriminated, or the migration pattern reading accuracy becomes low. Low accuracy of the migration pattern reading results in low reliability of the obtained information on the base sequence in nucleic acid or the like.